Introducing the Microbiome Dr Ailsa Hart Director IBD Unit, Consultant Gastroenterologist, St Mark’s Honorary Senior Clinical Lecturer, Imperial College

Introduction What is the gut microbiome? How does the gut microbiome play a role in disease? - role in inflammatory bowel disease How can we modulate the gut microbiota as a therapy - faecal transplant

The gut microbiota 1014 gut bacteria and 1013 cells in body Most densely populated ecosystem on Earth 4 major phyla (Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria) Provide traits we have not had to evolve on our own “Virtual” organ Genes in gut flora 100 times our own genome Mostly anaerobic, steep stomach acid driven proximal distal gradient

Function of gut microbiota Germ-free animals Exist and survive But abnormalities of: Immune function (oral tolerance) Metabolic function (altered enzymes) Physiological function (altered motility) Trophic function (altered cell turnover) What is the function of the gut flora – well much can be deduced from studies in germ free animals – These animals exist and survive indicating that the gut flora is not a prerequisite for life but the animals do have profound abnormalities of immune function – reduced GALT, small PP, dec LP AND defective CMI and induction of oral tolerance. In addition these animals have abnormalities of…

Host – microbiota interactions Function of gut microbiota Host – microbiota interactions Hooper et al. Science 2001 A single commensal bacteria switches on genes involving Mucosal barrier function Nutrient absorption/ dietary energy extraction Enteric nervous system Intestinal maturation Immune system development One of the most illuminating studies assessing the function of the gut flora is this one by Hooper et al. reported in Science in 2001. This group introduced a single commensal bacteria into a germ free animal and assessed by gene array analysis the genes which were switched on. They found that a multitude of genes were switched on including …… Although clearly a reductionist model it does highlight the huge potential of the gut flora in its entirity to interact with and influence host physiology.

The Meta HIT project

The Human Microbiome Project

The human microbiome project 5 year project 242 healthy men/women - samples from different body sites 10,000 different types of organism found Is there a core set of microbes that all humans share? Diversity of microbes across human beings Diet, host genetics, early microbial exposure Unique communities of microbes at different body sites At specific body sites, many microbes had similar genes/functions Challenges thinking of one-microbe model of disease More likely “function” of group of microbes changes

Gut microbiota “pathology” e.g. inflammatory bowel diseases functional bowel disease gastrointestinal infections non-GI diseases - obesity, metabolic syndrome, atopy/allergy

Genetics of IBD >160 independent IBD susceptibility loci 2001 2007 2008 2011 2012 For many loci fine mapping is underway as part of the Immunochip experiment

What has genetics taught us about IBD? > 75,000 cases & controls Information about pathways involved in disease process Over 2/3 of genes are shared between UC &CD 30 CD-specific and 23 UC-specific Particular overlap between ankylosing spondylitis and psoriasis 1Jostins et al Nature 491 (7422):119-124; 2Lees et al Gut 2011;60:1739-53 (diagram)

Experimental models of colitis Germ-Free No Colitis Animal models of colitis What is the evidence that the gut flora drives the inflammation in IBD. The best evidence comes from animal models

Experimental models of colitis Germ-Free No Colitis Animal models of colitis Bacterial Colonisation Colitis

In humans… Faecal stream diversion alleviates Crohn’s Reanastomosis triggers recurrence Infusion of luminal contents into excluded normal bowel induces inflammation

What part of the gut microbiota drives inflammation? Single organism? Expansion or relative contraction? “Functional” changes?

Reduced diversity of faecal microbiota In CD

Reduced Firmicutes in Crohn’s disease

- Reduction of a major member of Firmicutes, F - Reduction of a major member of Firmicutes, F. prausnitzii, associated with higher risk of postoperative recurrence of ileal CD. Experimental replacement of F. prausnitzii had anti-inflammatory effects

But changes in “function” of microbiota…. Mucosa/stool samples from 231 IBD patients & controls 16S gene pyrosequencing/shotgun metagenomics Major shifts in oxidative stress pathways Decreased carbohydrate metabolism Decreased amino acid synthesis In ileal Crohn’s, increases in virulence and secretion pathways Overall CD is not caused by diminished diversity alone – but requires a susceptible genotype – as confirmed by research in mice with human relevant susceptibility mutations Mucin, which is rich in cysteine and glycosylated sugars, is abundant in the intestinal epithelium, and it is upregulated during inflammation. The increases in cysteine metabolism and N-acetylgalactosamine transporters may reflect a shift in the microbiome towards greater abundance of microbes that use mucin as a primary energy source (Figure 6). This functionality suggests activity at the mucosa and this may be problematic for a damaged IBD epithelium with compromised barrier function. This raises the interesting possibility that E. coli or related species in IBD may be highly represented because they gain a competitive advantage from oxidative stress and are better able to compensate for it with glutathione production. Microbial function more consistently altered than microbial composition Morgan et al. Genome Biology Sept 2012

Challenges Clinical phenotype confounders “healthy” controls Sampling age, gender, smoking ethnicity, diet, surgery medications “healthy” controls Sampling faeces v mucosa axial and longitudinal variation replication multiple samples from same region longitudinal sampling Communication clinicians microbial ecologists bioinformatics statisticians Technical 16S sequence metagenomics metatranscriptomics metabonomics … economics

Can the gut microbiota be modified? If so, how?

Faecal transplantation - history 1600s – “transfaunation” – gastric contents transplanted into animals unable to ruminate Bedouin of northern Africa – “coprphagia” – ingestion of camel dung as treatment for dysentery WWII – same practice in soldiers – led to Bacillus subtilis as probiotic Faecal transplant (FT ) - treatment in humans for pseudomembranous colitis in1958 Landy et al. Aliment Pharmacol Ther 2011; 34: 409-15

Publications on faecal transplantation

Faecal transplantation in C. difficile Over 376 cases of faecal transplant (FT) for C. diff infection 17/22 studies of FT are in refractory/fulminant C. diff Enemas; via duodenal tubes; via colonoscopes Overall success rates of >90% Landy et al. Aliment Pharmacol Ther 2011; 34: 409-15

Faecal transplantation in C. difficile First randomised controlled trial Aimed to recruit 120 patients – stopped early 13/16 (81%) in FT group – resolution of diarrhoea 4/13 (31%) in vancomycin group 3/13 (23%) in vancomycin + bowel lavage After FT, ↑diversity (similar to healthy donors) Van Nood et al. NEJM January 2013

Faecal transplantation in IBD 41 patients with IBD (27 UC; 12 CD; 2 indeterminate) Majority (19/25) had reduction in symptoms/ 15/24 remission Resolution of C. diff infection in 15/15 Damman et al. Am J Gastro. 2012; 107: 1451-9; Anderson et al. APT Sept 2012

with faecal transplantation may be effective in refractory pouchitis Faecal transplantation in pouchitis Hypothesis – increasing diversity and altering selection of resistant bacteria with faecal transplantation may be effective in refractory pouchitis Pilot study of faecal transplantation in patients with chronic pouchitis Microbiological; immunological; histological & clinical assessment pre and post transplant

Faecal transplantation in pouchitis PDAI CGQoL score pouch frequency No major adverse events; 3 patients reported adverse effects nausea (n=3); bloating (n=2) Landy et al. ECCO 2013

Unresolved issues… Can microbiota be altered in IBD with FT? If so, does genetic pressure / indigenous bacteria affect ability to change? Donor - mixture of phylogenetically diverse bacteria – which? Frequency and route of administration? Use of concomitant treatment? Fresh versus frozen (banks)? Safety?

Altering microbiota - beyond IBD ….. Other gastrointestinal disorders Functional bowel disorders Gastrointestinal infections e.g. Clostridium difficile, rotavirus Non-intestinal disorders Atopic diseases Type I diabetes/ metabolic syndrome Obesity ……

Obesity

Could manipulation of gut microbiota influence obesity? Calorie extraction from food varies depending on gut flora “Obese flora” more effective at extracting energy from food Could manipulation of gut microbiota influence obesity?

Could manipulation of gut microbiota influence malnutrition? Calorie extraction from food varies depending on gut flora “Obese flora” more effective at extracting energy from food Could manipulation of gut microbiota influence malnutrition?

Summary Gut microbiota – virtual organ; microbiome; metobonome Role of microbiota in IBD Modulation of gut flora as a therapeutic intervention Mechanistic approach Far reaching potential in GI (and non-GI) disease